OPA1: How much do we know to approach therapy?

G Model

ARTICLE IN PRESS

YPHRS-3830; No. of Pages 12

Pharmacological Research xxx (2018) xxx–xxx

Contents lists available at ScienceDirect

Pharmacological Research journal homepage: www.elsevier.com/locate/yphrs

Invited Review-pharmacology across disciplines

OPA1: How much do we know to approach therapy? Valentina Del Dotto a , Mario Fogazza b , Guy Lenaers c , Michela Rugolo b , Valerio Carelli a,d,∗∗ , Claudia Zanna b,∗ a

Unit of Neurology, Department of Biomedical and NeuroMotor Sciences (DIBINEM), University of Bologna, Bologna, Italy Department of Pharmacy and Biotechnology (FABIT), University of Bologna, Bologna, Italy MitoLab team, CNRS UMR6015, INSERM U1083, Institut MitoVasc, Université d’Angers, 49933 Angers cedex 9, France d IRCCS Institute of Neurological Sciences of Bologna, Bellaria Hospital, Bologna, Italy b c

a r t i c l e

i n f o

Article history: Received 20 December 2017 Received in revised form 12 February 2018 Accepted 12 February 2018 Available online xxx Keywords: OPA1 mutations Dominant optic atrophy Mitochondria dynamics DOA therapy

a b s t r a c t OPA1 is a GTPase that controls several functions, such as mitochondrial dynamics and energetics, mtDNA maintenance and cristae integrity. In the last years, there have been described other cellular pathways and mechanisms involving OPA1 directly or through its interaction. All this new information, by implementing our knowledge on OPA1 is instrumental to elucidating the pathogenic mechanisms of OPA1 mutations. Indeed, these are associated with dominant optic atrophy (DOA), one of the most common inherited optic neuropathies, and with an increasing number of heterogeneous neurodegenerative disorders. In this review, we overview all recent findings on OPA1 protein functions, on its dysfunction and related clinical phenotypes, focusing on the current therapeutic options and future perspectives to treat DOA and the other associated neurological disorders due to OPA1 mutations. © 2018 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 1.1. OPA1 gene, protein and mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Clinical phenotypes associated to OPA1 mutations (dominant and recessive) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 OPA1 functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.1. Overview of OPA1 mitochondrial functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.1.1. The fusion process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.1.2. The cristae structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.1.3. The mtDNA stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.1.4. The bioenergetic competence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.2. Other OPA1- dependent processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.2.1. Cellular redox homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.2.2. Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.2.3. Mitochondrial quality control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.2.4. Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.2.5. Extra-mitochondrial OPA1 function? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.3. Is a specific mitochondrial function associated with a definite OPA1 isoform? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.3.1. Single vs multiple OPA1 isoforms functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.3.2. Is there a specific role for long and short forms? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.1. Gene therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

∗ Corresponding author at: Department of Pharmacy and Biotechnology (FABIT), University of Bologna, Bologna, Italy. ∗∗ Corresponding author at: Unit of Neurology, Department of Biomedical and NeuroMotor Sciences (DIBINEM), University of Bologna, Bologna, Italy. E-mail addresses: [email protected] (V. Carelli), [email protected] (C. Zanna). https://doi.org/10.1016/j.phrs.2018.02.018 1043-6618/© 2018 Elsevier Ltd. All rights reserved.

Please cite this article in press as: V. Del Dotto, et al., OPA1: How much do we know to approach therapy? Pharmacol Res (2018), https://doi.org/10.1016/j.phrs.2018.02.018

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4.1.1. Is OPA1 gene therapy feasible? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.1.2. Mutation-independent strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.1.3. CRISPR-Cas9 and gene editing strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.2. Drugs potentially able to complement OPA1 defective function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.2.1. Drugs targeting complex I and ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.2.2. Drugs modifying mitochondrial biogenesis and mitophagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction 1.1. OPA1 gene, protein and mutations The OPA1 gene, localized on 3q28, spans more than 100 kb and includes 31exons [1]. The alternative splicing of exons 4, 4b and 5b generates 8 different mRNA variants coding 8 isoforms, variably expressed in the different human tissues, witnessing a fine regulation of OPA1 mRNAs. All OPA1 isoforms are ubiquitously expressed, nevertheless splicing variants containing exon 4 are consistently more represented [2]. The exon 4 is evolutionarily conserved, while the exons 4b and 5b are both specific to vertebrates [2]. The eight OPA1 mRNA variants encode proteins of 924–1014 aminoacids that include at the N-terminal region a mitochondrial targeting sequence (MTS) followed by a first transmembrane domain (TM), anchoring the protein to the inner mitochondrial membrane (IMM), and the three alternate spliced domains corresponding to exons 4, 4b and 5b [2]. Exon 4 domain does not present any remarkable feature, whereas the domains corresponding to exon 4b and 5b provide two additional hydrophobic domains, TM2a and TM2b. 5b exon encodes also a coiled coil domain (CC0). The following part of the protein includes a coiled coil domain (CC1), then the conserved dynamin regions: the GTPase domain, the middle domain with unknown function and the C-terminus GTPase effector domain (GED) also containing a coiled coil domain (CC2) [2,3]. After the import of OPA1 precursors through the mitochondrial membranes, the cleavage of the MTS by the mitochondrial processing peptidase (MPP) generates the membrane-anchored long forms (l-forms). OPA1 may be further processed downstream the TM region at the S1 and S2 cleavage sites, located on domains 5 and 5b respectively, producing soluble short forms (s-forms) that can be peripherally attached to the IMM or diffuse in the inter-membrane space (IMS) and associate to the outer mitochondrial membrane (OMM) [4]. The proteolytic cleavage is carried out by two IMM peptidases, the ATP-dependent protease YME1L [5,6] and the zinc metalloprotease OMA1 [7], whose activities set a nearly equimolar equilibrium of l- and s-OPA1 forms under basal conditions. Finally, the four isoforms containing the exon 4b are totally processed into s-forms [5], generating a short peptide of 10 kDa, including TM1 and TM2a, required for anchoring to the IMM the mitochondrial DNA (mtDNA) nucleoids [8], multiple intra-matrix structures usually constituted by a single-copy mitogenome packaged by the mitochondrial transcription factor A (TFAM) [9]. OPA1 has been identified as the first gene involved in Dominant Optic Atrophy [10,11]. Since this discovery, novel OPA1 mutations are continuously reported on the locus-specific database dedicated to OPA1 (http://opa1.mitodyn.org/; [12]). From the 414 OPA1 variants actually listed, more than 60% are considered pathogenic and two-thirds of them are in the coding sequence, mainly located in the dynamin and GTPase domains. Most OPA1 mutations are substitutions (290) or deletions (94), whereas only few duplications (20), insertions (5) and in/del (5) mutations have been annotated. Mutations are mostly family-specific, but some of them are recurrent. In about 50% of cases the pathogenic mutations introduce a premature

stop codon [12], leading to the consequent truncation of the open reading frame, which undergoes mRNA decay, ultimately determining the loss of function of the mutant allele. Thus, these variants share haploinsufficiency as the main pathological mechanism [13]. 2. Clinical phenotypes associated to OPA1 mutations (dominant and recessive) In 1959 Poul Kjer described a dominantly inherited form of bilateral optic neuropathy (DOA), a blinding disorder characterized by the isolated degeneration of retinal ganglion cells (RGCs) and the atrophy of optic nerve [14]. A syndromic phenotype of DOA, including extraocular features such as deafness and chronic progressive external ophthalmoplegia (CPEO) was also described in two different families in the US and in Europe [15,16]. Only in the year 2000 a large proportion of DOA families was associated with mutations in the OPA1 gene, allowing for the identification of the molecular cause [10,11]. DOA has recently been estimated to have a prevalence of 1 in 25,000 and is currently considered the most common mitochondrial inherited optic neuropathy [17]. Since 2003 cases of DOA associated with sensorineural deafness were increasingly recognized as due to a specific missense mutation affecting the GTPase domain of OPA1, the p.Arg445His [18,19]. This same mutation was also found in both syndromic DOA families described in the 80s, which besides deafness also had CPEO [20]. Multiple labs since 2004 also observed a dominant multisystemic disorder affecting multiple families characterized by extraocular features emerging after the occurrence of optic neuropathy, which included sensorineural deafness, ataxia, CPEO with mitochondrial myopathy and peripheral neuropathy. These families with a syndromic form of DOA, which resembled the two families carrying the p.Arg445His OPA1 mutation [15,16,20], were found to bearing either this or other mutations affecting the GTPase domain of OPA1 and this clinical entity was named DOA “plus” [21,22]. In the subsequent years, OPA1 mutations have been linked to an expanding spectrum of neurodegenerative phenotypes, such as spastic paraplegia [23], multiple sclerosis-like syndrome [23–25], Behr-like syndrome [26] and most recently syndromic parkinsonism and dementia [27,28]. Furthermore, besides monoallelic dominant mutations, there has been an increasing recognition of biallelic mutations associated with complex and severe phenotypes. In fact, since 2011 emerged many reports of OPA1 compound heterozygosity, in cases with early-onset Behr syndrome [29–32], characterized by optic neuropathy and visual impairment in the first years of life, further complicated by spinocerebellar degeneration, pyramidal signs, peripheral neuropathy, gastrointestinal dysmotility and retarded development. Multiple unrelated patients were reported to harbor the same hypomorphic missense variant p.Ile437Met combined with a second clear-cut pathogenic mutation [29,30,32,33]. This hypomorphic mutation has an incidence of 1/300 in the western population, and by itself has no relevant consequence on health [30]. Furthermore, novel compound heterozygous OPA1 mutations have been identified in patients with optic atrophy, sensorimotor neuropathy and congenital cataracts [34], as well as severe



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Fig. 1. Diagram of the clinical and molecular OPA1-related disorders. DOA, first described in 1959 by Kjer, has been associated with OPA1 gene only in 2000. DOA and neurosensory deafness (DOAD) have been described in patients with the R445H mutation in OPA1 in 2003. Successively, in 2004 and 2005 the same mutation was identified as responsible also for the DOA+ syndromic disorder, initially reported in 1984 and 1985 by Treft and Meire. Since 2008, when OPA1 missense mutations have been linked to mitochondrial myopathy with COX-deficient fibers and to multiple deletions of mtDNA, a heterogenic ramification has developed for OPA1-associated neurodegenerative disorders, involving monoallelic dominant mutations (pink) and biallelic mutations (blue). The figure was prepared following the guidelines for colorblind [139].

Leigh-like encephalopathy [33,35], highlighting a growing occurrence of truly recessive cases. Furthermore, two unrelated patients with homozygous OPA1 recessive mutations have been recently reported. The p.Ala394Thr change induced early onset progressive spastic ataxia and sensory motor polyneuropathy, in the absence of optic atrophy [33], whereas the p.Leu534Arg change promoted a fatal, infantile, encephalopathy with progressive hypertrophic cardiomyopathy [36] (see Fig. 1 for a chronological representation of the clinical phenotypes of OPA1 related disorders). Strikingly, in most DOA “plus” cases, but also in non-syndromic DOA, carrying heterozygous mutations mostly affecting the GTPase domain the accumulation of mtDNA multiple deletions was documented in post-mitotic tissues, which in skeletal muscle biopsies is phenotypically exemplified by the occurrence of cytochrome c oxidase (COX)-deficient and ragged red fibers [21,22]. Essentially, shorter mtDNA molecules lacking portions of the mitogenome are stochastically generated during a defective replicative process, which seems enhanced by the OPA1 missense mutations; these partially deleted molecules of various sizes may prevail over the wild-type mtDNAs in specific domains of muscle fibers, leading to focally defective respiratory chain activity. This observation led for the first time to the concept that mitochondrial fusion is a key mechanism for mtDNA maintenance in humans, while adding a pathogenic mechanism for these complex multisystemic disorders. Remarkably, more recently mtDNA depletion was also observed in the severe recessive phenotypes [36], further highlighting the role of OPA1 and mitochondrial fusion in keeping mtDNA integrity.

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Fig. 2. Schematic representation of OPA1 s direct interactors, identified by coimmunoprecipitation. Interactors involved in mitochondrial dynamics (yellow): mitofusin 1 and 2 (MFN1, MFN2), Williams-Beuren syndrome “critical region”16 (WBSCR16) and leucinerich repeat kinase 2 (LRRK2); in mtDNA maintenance (orange): mitochondrial transcription factor A (TFAM) and DNA polymerase gamma (POL␥); in mitochondrial quality control (pink): sirtuins 3 and 4 (SIRT3, SIRT4), FUN14 domain containing 1 (FUNDC1) and BCL2 interacting protein 3 (BNIP3); in apoptosis (blue green): HtrA serine peptidase 2 (OMI/HTRA2), HIG1 hypoxia inducible domain family member 1A (HIGD-1a), apoptosis inducing factor (AIF) and presenilin-associated rhomboidlike (PARL); in cristae integrity (blue): solute carrier family 25 (SLC25A), the reactive oxygen species modulator 1 (ROMO1), coiled-coil-helix-coiled-coil-helix domain containing 6 (CHCHD6) and Mitofilin; and in energetics (sky blue): respiratory chain supercomplexes (RCS) and nucleoside diphosphate kinase D (NDPK-D) are reported. The figure was prepared following the guidelines for colorblind [139]

The association of OPA1 mutations with defective mtDNA maintenance matched similar phenotypes associated with MFN2 mutation [37,38]. 3. OPA1 functions 3.1. Overview of OPA1 mitochondrial functions A large body of studies using different cell models including OPA1 silencing and/or stable genetic ablation, as well as cells derived from DOA patients bearing OPA1 mutations, led to defining OPA1 as a multifunctional protein able to interact with several partners (Fig. 2). 3.1.1. The fusion process At first, OPA1 was shown to mediate the fusion at the IMM [39]. OPA1 is now recognized as a key player of mitochondrial network dynamics operating in close collaboration with the other dynamins-like GTPases as the pro-fusion Mitofusins, MFN1 and MFN2, as direct interactors [40], and the pro-fission DRP1 and DNM2 [41]. It has been reported a direct interaction of OPA1 with the leucine-rich repeat kinase 2 (LRRK2), mutated in Parkinson disease, and with WBSCR16, a guanine nucleotide exchange factor (GEF) OPA1-specific, both proposed as additional players in the mitochondrial dynamics [42,43]. The mechanism of OPA1-mediated fusion process at the IMM and interactions with MFNs in the OMM have been extensively discussed elsewhere [44,45]. In the following section 3.3.1, the very hot topic concerning the contribution of l- and s-OPA1 forms to fusion will be addressed.



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3.1.2. The cristae structure The cristae junctions are kept tight by oligomerization of proper amounts of soluble and membrane-bound forms of OPA1 [46] and the lack of OPA1 or the presence of OPA1 pathogenic mutations is associated with dramatic disorganization of the mitochondrial ultrastructure [39,47]. The dynamic behavior of mitochondrial ultrastructure and the complexity of proteins/complexes interactions involved in its maintenance are important issues recently deeply investigated. Special focus points to the mitochondrial contact sites and cristae organizing system (MICOS), a large oligomeric complex strategically located at cristae junctions that stabilizes membrane curvature and forms contacts between the IMM and OMM [48]. Upon deletion of MICOS core subunits, crista junctions collapse and OPA1 levels are reduced, suggesting that MICOS complex links IMM architecture to mitochondrial network dynamics [49]. Remarkably, two of the MICOS subunits (Mic60/mitofilin and Mic25/CHCHD6) were shown to directly interact with OPA1 [50]. Furthermore, OPA1 is epistatic to Mic60/mitofilin in the regulation of cristae junction number and stability, suggesting the GTPase as the exclusive regulator of cristae junctions width [51]. Finally, OPA1 interacts also with a number of mitochondrial SLC25A transporters, which sense variation in energy substrate availability, and the reactive oxygen species modulator 1 (ROMO1), both regulating OPA1 oligomerization to modulate cristae width and assembly of the ATP synthase [52,53]. Thus, the maintenance of cristae structure entails a large array of protein interactions, where OPA1 acts at the IMM in close contact with scaffolding MICOS subunits as well as with OMM components like Sam50. This central protein hub operates in functional relationship also with phospholipids such as phosphatidylethanolamine and cardiolipin, thus influencing the properties of the IMM, its curvature and fusion [54]. It is easy to envisage that future investigations on this hub of protein/protein and protein/lipids interactions will provide important clues to shed light on these aspects. 3.1.3. The mtDNA stability The maintenance of the mitochondrial genome is strictly linked to OPA1, since its genetic ablation causes a marked mtDNA depletion [55] and the presence of a defined class of OPA1 mutations, as reported above, produces mtDNA instability with accumulation of multiple deletions [21,22] and, more rarely, mtDNA depletion [36]. Considering the evidence that similar consequences occur also with lack or mutations of MFNs, it follows that the mitochondrial network fusion with constitutive mixing of matrix contents is crucial to preserve the integrity of mitochondrial genome. By silencing each of the three alternative exons in HeLa cells, it turned out that only exon 4b silenced cells displayed mtDNA depletion and a marked alteration of nucleoids distribution throughout the network [8]. Furthermore, a peptide encompassing the OPA1 N-terminus and the domain corresponding to exon 4b was shown to directly interact with TFAM, POL␥ and mtDNA, confirming that exon 4b containing OPA1 variants may contribute to mtDNA stability by nucleoid attachment to the IMM [8]. 3.1.4. The bioenergetic competence OPA1 depletion was associated with a severe bioenergetic perturbation, characterized by reduced oxygen consumption rate and ATP levels, loss of respiratory supercomplexes organization and incomplete assembly of complex V [52,56,57]. As a consequence of the energetic dysfunction, mitochondrial Ca2+ uptake and Ca2+ retention capacity were also reduced, impairing the Ca2+ homeostasis and further worsening the cellular phenotype [58,59]. Analysis of OPA1 mutated fibroblasts also disclosed a mild but significant energetic dysfunction, mostly affecting complex I [47] and

complex IV [60]. Immunoprecipitation experiments showed that OPA1 physically interacts with subunits of the respiratory complexes, suggesting a direct role in the stabilization of the overall respiratory chain [47,60,61]. This issue has been further investigated in the mouse model of Opa1 conditional ablation, where mtDNA is normal, unraveling that the cristae shape governs the supramolecular organization of respiratory supercomplexes and hence the oxidative phosphorylation efficiency [62]. In 2013, the interaction between OPA1 and the nucleoside diphosphate kinase Nm23-H4/NDPK-D, a complex likely involved in phosphotransfer activity, has been reported [63]. Finally, OPA1 was also shown to be required for transient matrix alkalinisation, renamed “mitopHflashes”, which preserves the ability of mitochondria to convert energy during drops in membrane potential (ym ) [64,65]. 3.2. Other OPA1- dependent processes 3.2.1. Cellular redox homeostasis The respiratory chain constitutes the main source of reactive oxygen species (ROS) within the cell. Considering the role of OPA1 in supercomplexes organization and shaping cristae structure, it is predictable that OPA1 dysfunction may imply ROS over-production and unbalanced redox homeostasis. Indeed, increased ROS levels were reported in a Drosophila model with homozygous or heterozygous mutations of Opa1 [66,67] and in cardiomyocytes from a Opa1+/− mouse models [68]. Furthermore, in brain cortex from Opa1+/− transgenic mouse, as well as in OPA1 down-regulated cortical neurons, a reduction of aconitase activity was reported, strongly supporting an unbalance in redox homeostasis. This is also confirmed by activation of the nuclear factor erythroid-derived 2 like 2 (NRF2) pathway, leading to increased levels of mitochondrial antioxidant defenses (i.e. superoxide dismutases 1 and 2, SOD1 and SOD2) [69]. Increased ROS leves were also observed in lymphoblastoid cells bearing OPA1 mutations [70] and altered levels of antioxidant enzumes were revealed in fibroblasts from patients with DOA, further supporting a perturbation of mitochondrial redox state [69]. Conversely, overexpression of OPA1 reduced mitochondrial ROS production by stabilizing supercomplexes [71]. Finally, a mutual relationship between OPA1 and ROS has been recently highlighted in cardiomyocytes, where increased ROS generation caused by overexpressing long-chain acyl-CoA synthetase 1 (ACSL1) altered the proteolytic processing of OPA1[72]. 3.2.2. Apoptosis The link between mitochondrial dynamics and apoptosis is well known and has been extensively discussed elsewhere [73,74]. We will here focus on the involvement of OPA1 in apoptosis, which became apparent since the first experiments of OPA1 silencing in HeLa cells, where the network fragmentation and the dramatic disruption of cristae structure were associated with release of cytochrome c from the OMM triggering caspase-dependent apoptotic nuclear events [39]. Interestingly, the anti-apoptotic function of OPA1 was found to be independent of its fusogenic function [75]. Predisposition to apoptosis was confirmed also in OPA1 mutant fibroblasts [47,76]. Accordingly, OPA1 overexpression protected cells from apoptosis by keeping tight the cristae and, therefore, by compartmentalization of cytochrome c and other pro-apoptotic factors within the cristae volume, thanks to the assembly of OPA1 oligomers [46]. Several studies have investigated the molecular mechanisms through which OPA1 can perform its anti-apoptotic function. BNIP3, a pro-apoptotic BH3-only protein of the Bcl-2 family, was shown to directly bind OPA1 and this association causes destruction of OPA1 oligomers, promoting the onset of apoptosis [77]. In normal conditions the serine protease Omi/Htra2 exerts a cyto-protective effect by direct binding to OPA1, whereas during apoptosis it is




released from IMS into the cytosol. Loss of Omi/Htra2 induces dismantling of cristae structure, and up-regulation of mild detergent extractable OPA1 protein. Accordingly, Omi/HtrA2 was proposed to impact on the IMM structure by modulating the localization of OPA1 to the cristae junctions [78]. Hypoxia-Induced Gene Domain protein-1a (Higd-1a), a small inner membrane protein, when overexpressed delays apoptosis by inhibiting the release of cytochrome c and reducing caspase activities [79]. Higd-1a was shown to bind OPA1 and to push the balance toward the long forms, protecting them from cleavage and promoting network fusion [80]. Finally, the interaction between OPA1and the IMM protease PARL (Presenilinassociated rhomboid-like), acting together in an anti-apoptotic pathway [81], and between OPA1 and AIF (Apoptosis inducing factor) [47] have also been reported. 3.2.3. Mitochondrial quality control As a central player of mitochondrial dynamics OPA1 takes also part to the mitochondrial quality control system, a complex process in which dysfunctional mitochondria are selectively degraded by mitophagy [82,83], through different pathways. These may involve the serine/threonine kinase PINK1 and the E3 ubiquitin ligase Parkin [84] or other mitophagy receptors such as BNIP3 [85] and FUNDC1 [86], which mediate interaction with LC3 and recruitment to the autophagy machinery. Over-expression of OPA1 inhibited mitophagy [87]; similarly the elongation of mitochondrial network by DRP1 inhibition protected from autophagic degradation during starvation [88]. In a recent in vitro neuronal model of DOA characterized by OPA1 haploinsufficieny, BNIP3 is downregulated leading to a reduced number of autophagosomes and of mitochondria within autophagosomes, both these effects being rescued by increasing the BNIP3 levels [89]. The two-faced role of BNIP3 acting as a pro-apoptotic factor and pro-autophagic/mitophagic element remains however to be explored. On the other hand, fibroblasts with OPA1 missense mutations in the GTPase domain, exhibiting a reduced OPA1 protein level and a highly fragmented mitochondrial network, were shown to present a constitutively activated basal mitophagy that could not be further increased by known mitophagic stimuli [27]. Similar results have been also reported in biallelic OPA1 mutations [90]. Kane and colleagues showed a genotype–phenotype correlation, associating missense mutations with increased autophagy and mitophagy, whereas haploinsufficiency mutations were linked to a reduced mitochondrial turnover and autophagy [91]. On the contrary, increased autophagy, mitophagy and vacuolated mitochondria have been observed in two Opa1+/− mouse models, particularly in the RGC layer or in the glycolytic muscle [92,93]. Further studies are needed to understand the molecular mechanisms causing these two different phenotypes, focusing also on the methods employed for quantitative mitophagy assessment. FUNDC1 is a protein inserted in the OMM that interacts with both DRP1 outside and OPA1 inside in a “see-saw model” of interaction, promoting mitochondrial fission or fusion, respectively. Under normal condition, the FUNDC1/OPA1 complexes are present in the IMS, whereas under mitochondrial stress conditions these complexes are dismantled and FUNDC1 recruits DRP1 from the cytosol to the mitochondria. Interestingly, the phosphorylation status of FUNDC1 dictates the interaction with DRP1 or OPA1 and with the LC3, thus serving as an “inside-out” mechanism coupling the stress responses to mitochondrial dynamics and quality control across the OMM [94]. New players in the mitochondrial quality control linked to OPA1 are also the three mitochondrial sirtuins SIRT3, SIRT4 and SIRT5, that are NAD-dependent protein deacetylases, all involved in metabolic homeostasis. SIRT3 directly binds OPA1 and promotes deacetylation of two lysine residues in the GED domain,

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thus increasing the GTPase activity and fusion efficiency [95]. In the case of SIRT4, its up-regulation was shown to reduce the network fragmentation by inhibiting DRP1 activity [96] and by increasing the ratio of l-/s-forms of OPA1, thus counteracting fission and mitophagy [97]. Mechanistically, the increase in the l-forms, as well as the direct interaction of OPA1 with SIRT4, suggest a new axis in mitochondrial quality control, where SIRT4 stabilizes the OPA1 l-forms, either by (in)direct protein–protein interaction or by protection from OMA1 mediated processing [97]. Also SIRT5 exhibits a protective effect against mitophagy when overexpressed, leading to increased levels of MFN2 and OPA1, thus preventing fragmentation, whereas the opposite effect was observed after SIRT5 silencing [98]. In summary, all three mitochondrial SIRTs shift the fusion/fission cycle toward fusion via OPA1, contributing to attenuate mitophagic clearance of dysfunctional mitochondria leading to their accumulation, which is characteristic of senescent cells and may be relevant for aging. 3.2.4. Aging It is well established that mitochondrial dysfunction occurs during the aging process [99]. The maintenance of mitochondrial network dynamics is particularly relevant for post-mitotic cells that do not divide, such as neurons and cardiac and skeletal muscles that use the fusion/fission machinery to preserve or restore the mitochondrial function. A failure of these systems predisposes to tissue dysfunction and degeneration. The finding that aged Opa1+/− mice display selective loss of glutamatergic, but not GABAergic, synaptic sites leading to dendritic degeneration suggests that OPA1 may be also relevant in the aging process [100]. Furthermore, in adult mice, muscle-specific knock-out of Opa1 induces precocious senescence and premature death. Particularly, ER stress caused by Opa1 deletion activates a signaling pathway to the nucleus via the unfolded protein response (UPR) and FoxOs, inducing a catabolic program of muscle loss and systemic aging [101]. A recent study reported a higher frequency of mitochondrial network fusion in human fibroblasts from old compared to young healthy donors, which indeed exhibited a significant shift toward mitochondrial fission. The increased content of OPA1 and MFN1 in old fibroblasts nicely correlated with enhanced mitochondrial fusion, which was shown to be strictly associated with metabolic reprogramming from glycolysis to oxidative metabolism [102]. Intervention of metabolic reprogramming during aging may extend healthy lifespan and minimize age-related health issues. 3.2.5. Extra-mitochondrial OPA1 function? An unpredicted extra-mitochondrial role for OPA1 has been reported in the lipid droplets of adipocytes, where OPA1 was shown to operate as an A-kinase anchoring protein (AKAP) for perilipin to promote lipolysis [103]. Lipid droplets represent a scaffold, containing also a number of proteins, for storage of neutral lipids, when fatty acids and cholesterol are in excess. What may be the function of the GTPase in this compartment remains unclear, although altered phospholipids amount has been reported in optic nerve and plasma of a Opa1+/− mouse model [104]. Further studies are required to define the extracellular OPA1 function in lipid metabolism. 3.3. Is a specific mitochondrial function associated with a definite OPA1 isoform? 3.3.1. Single vs multiple OPA1 isoforms functions Considering that multiple mitochondrial functions are associated with OPA1, it was reasonable to ask whether these functions could in some way be related to the multiplicity of isoforms expressed in the different human tissues. By selective silencing each of the three alternative exons in HeLa cells, it was found



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as cycloheximide treatment or nutrient starvation, l-form cells become significantly more filamentous [56,57,107]. These observations suggest the presence of unknown regulatory mechanisms that control the activity of OPA1 l-form in vivo. Accordingly, it has to be highlighted that network morphology and fusion are not always synonymous concepts in mitochondrial dynamics (Fig. 3). On the contrary, the s-form is insufficient to maintain a filamentous mitochondrial network [56,57,108]. The in vitro membrane fusion assay indicated that s-forms alone are not sufficient to promote fusion, but their addition increase the l-form-dependent fusion activity, suggesting a role as coadjutor in the fusion process. The artificially membrane-anchored s-form shows a minimal capacity to stimulate fusion [106], in agreement with the minimal, but significant, fusion activity of s-form expressed in Opa1−/− MEFs [57]. It may be that the increased unbalance towards s-forms is a cellular mechanism to reduce fusion, shifting the equilibrium to fission. Fig. 3. Summary of OPA1 isoforms functions. Each of the eight OPA1 isoforms is able to maintain the mtDNA content, build the cristae and organize the functional respirasome. L-forms alone support mitochondrial fusion and s-forms alone preserve energetics, both independently of mitochondrial morphology. Thus, fusion and morphology are not equivalent and should be considered as distinct. The figure was prepared following the guidelines for colorblind [139].

that exon 4-containing isoforms were involved in maintenance of m and mitochondrial fusion, whereas silencing of exon 4band 5b-containing isoforms induced typical hallmarks of apoptosis [2]. Furthermore, exon 4b-containing isoforms were involved in mtDNA maintenance [8]. Through use of this exon-specific silencing approach, however, it was not possible to evaluate whether a single OPA1 isoform is specifically associated with a definite mitochondrial function. Recently, each of the eight OPA1 splice forms was stably expressed alone in Opa1-null cells, proving that expression of any OPA1 isoform was able to preserve the physiological level of mtDNA, to shape the cristae, and to properly assemble the respiratory supercomplexes, as well as complex V, that were severely impaired in Opa1−/− MEFs [57] (Fig. 3). Interestingly, the almost complete fragmentation of mitochondrial network of Opa1−/− MEFs was only partially recovered by mRNA splice forms generating both l- and s-forms, in agreement with a previous report [5], and a specific balance of l- and s-forms and an adequate amount of the protein was required for full recovery [57]. The plethora of OPA1 isoforms may provide to the cell the elasticity to modulate minimal adjustments able to adapt at the fluctuations of cellular metabolic and stress conditions. 3.3.2. Is there a specific role for long and short forms? As the consequence of the intricate OPA1 proteolytic processing, a typical pattern of l- and s-forms is generated in each cell system. Both forms were similarly effective in restoring the mtDNA content and in keeping the cristae density and cristae junction tightness [56,57]. The s-form alone was more efficient than the l-form in rescuing bioenergetics [57] or similar to the l-fom [56] (Fig. 3). The l-form alone was shown to be fusion competent, as demonstrated indirectly in the double Oma1 and Yme1 l knockout MEFs, presenting only l-forms [105]. This was confirmed by direct measurement of the mitochondrial fusion rate in Opa1−/− MEFs expressing an uncleavable isoform 1, revealing that it is as competent in fusion as a mixture of l- and s-forms [56,57]. In agreement, an in vitro membrane fusion assay demonstrated that the OPA1 l-form and cardiolipin are the minimal components sufficient and necessary for fusion [106]. It remains to be understood why l-forms, that exhibit an almost normal fusion activity when expressed in OPA1-null cells, are unable to display a normal mitochondrial network morphology, which indeed appears completely fragmented. However, when these cells are exposed to a stress condition, such

4. Therapy 4.1. Gene therapy 4.1.1. Is OPA1 gene therapy feasible? Gene therapy is strategically aimed at complementing the mutant gene by expressing the wild type allele, thus allowing disease treatment at the molecular level [109]. DOA, being a monogenic disease, is a good candidate for gene therapy, particularly for patients harboring OPA1 mutations leading to haploinsufficiency. Less clear is the scenario of missense mutations, where a dominant negative effect is assumed. The eye is an excellent organ to be targeted by gene therapy, as it is small, highly compartmentalized and easily accessible for the therapeutic intervention. Furthermore, the eyes can be efficiently monitored for evaluation of therapeutic efficacy by noninvasive approaches, such as electroretinography and optical coherence tomography, providing clear measurable endpoints for clinical trials [110,111]. In fact, a growing number of clinical trials are being developed to treat retinal genetic disorders by gene therapy. Encouraging results have been obtained by the Leber’s congenital amaurosis trial in humans, where an improvement in visual and retinal functions has been achieved a few months after treatment with a single unilateral intravitreal injection of adeno-associated virus (AAV) and maintained during the 3 years follow-up [112]. In fact, the results of this first randomized phase III gene therapy trial for a genetic disease (trial NCT00999609) showed improved light sensitivity and visual fields in these patients and the outcome of the follow-on phase I study suggested that these therapeutic effects might last at least 3 years [113,114]. More recently, the phase I/IIa safety trial (trial NCT02064569) with Leber’s hereditary optic neuropathy (LHON) also showed a potential treatment effect only in the patients with earlier onset vision loss, as compared with the more chronically affected patients (Uretsky 2017 Neurology April 18, 2017; 88 Supplement S26.005), supporting the strategy of the ongoing phase III trials NCT02652767 and NCT02652780. All these encouraging results cast hope to setting a therapy for OPA1-related DOA, possibly by using an AAV vector, given the tropism for RGCs. Indeed, the intraviteral injection of the AAV2 vector has proven efficacy and is relatively safe. The only adverse events are ascribed to the surgical procedure, which may lead to some inflammatory response [109,110,115]. Looking at the perspective of an OPA1-gene therapy, one first issue concerns the effect of a slight overexpression of OPA1, which has been recently explored in mouse models. A mild increase of isoform 1 protein level protects mice from denervationinduced mitochondrial dysfunction and muscular atrophy, from heart and brain damage induced by ischemia-reperfusion injury




and from Fas-induced liver apoptosis [71]. Moreover, increased OPA1 level efficiently ameliorates the phenotype of Ndufs4-/- and Cox15sm/sm mice, by improving cristae ultrastructure, complexes and RCSs organization and mitochondrial respiration [116]. A second issue to consider is which OPA1 isoform should be used in humans as the most efficient to rescue the defective clinical phenotype. Besides isoform 1 that has been used in mice, all eight isoforms, if slightly overexpressed in cells have comparable capability to recover mtDNA, cristae and energetics but not network dynamics [57]. Thus, the preferable isoform should comply tissuespecific requisites, such as to restore the appropriate OPA1 amount, as well as the balance between l-/s-forms. To this end it becomes pivotal to investigate the profile of OPA1 isoforms expression, OPA1 levels and l-/s-forms ratio in wild type and mutant human RGCs, to understand the specific alterations under pathological conditions. With the perspective to develop a personalized therapy specific for the patient, the isoform with the best therapeutic potential will be chosen based on the OPA1 alterations caused by the specific mutation type in that individual context. A third issue concerns the gene therapy applied to missense mutations, particularly those associated with the DOAD, DOA “plus” or Behr syndrome. Considering the assumption of a dominantnegative mode of action of these mutations, just increasing OPA1 levels may not be effective. A reduced amount of OPA1 protein in fibroblasts carrying some of these missense mutations has been shown [27,91], therefore we cannot exclude that increasing wildtype protein levels may ameliorate to some extent the clinical phenotype. It remains that for the severe multisystemic DOA “plus”, it will be necessary to develop a gene therapy for multiple tissue targets. Encouragingly, delivery strategies for different organs have been already developed [109]. Delivery to skeletal muscle is particularly interesting, given that myopathy is one of the most frequent clinical symptoms of DOA “plus”. Three OPA1 mouse models recapitulating the human pathology are available to evaluate safety and efficacy of gene therapy, harboring the truncative mutations in exon 8 (c.1051C > T), intron 10 (c.1065 + 5 G > A), and exon 27 (c.2708-2711delTTAG) [93,117,118]. A first pre-clinical trial is currently ongoing in the mouse model carrying the delTTAG mutation, based on the use of the wild-type isoform 1 (Lenaers Lab ongoing, See Note Added in Proof). On the other hand, it will be necessary to develop also a mouse model carrying missense mutations, to test the efficacy of OPA1 gene therapy in presence of a dominant-negative allele. 4.1.2. Mutation-independent strategies Eye diseases may present a limited therapeutic window for corrective gene therapy and over 240 genes are associated with retinal degeneration in humans. For these reasons, mutation-independent gene therapy strategies have been developed, allowing the treatment of different diseases, particularly when they are in advanced states of their natural history [110]. This approach may be considered also for DOA patients where RGCs and optic nerve are moderately or severely degenerated and, possibly, as alternative therapy for OPA1 missense mutations. Most strategies are developed to reduce RGCs degeneration, aimed at inhibiting the execution of cell death, for example by using the X-linked inhibitor of apoptosis (XIAP), others at increasing neuronal survival by using growth factors, as the ciliary neurotrophic factor (CNTF), the glial-cell derived neurotrophic factor (GDNF) and the brain-derived neurotrophic factor (BDNF), or by using enzymes and transcription factors that have an antioxidant final outcome, as NRF2 [110,119]. 4.1.3. CRISPR-Cas9 and gene editing strategies The discovery of the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and CRISPR-associated protein (Cas)

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system and its implementation in human cells [120,121] has opened the fascinating scenario for DNA editing in biomedicine. CRISPR/Cas system is an increasingly accurate and efficient tool to edit the human genome and it is particularly attractive for treating inherited non-syndromic conditions, particularly eye diseases [122,123]. The AAV2 delivery method containing the CRISPR-Cas system have been already developed and used in different types of human cells in vitro. The CRISPR-Cas strategy may be applied as a cell replacement therapy, as shown by trials that use iPSCderived ocular tissue after mutation correction, or as a gene editing approach of somatic cells, where the activity of AAV2-mediated CRISPR-Cas gene editing is applied to retinal cells in vivo [123,124]. This system has been used in a mouse model of retinitis pigmentosa to treat a missense mutation, showing retinal degeneration prevention and visual function improvement after a single sub-retinal injection [125]. Considering the DOA patients, this type of therapeutic approach may be the answer to the dominant missense mutations, in additions to those causing haploinsufficiency. Indeed, while OPA1 gene therapy may be useful in the case of haploinsufficiency alleles, the CRISPR-Cas technologies has the edge of precisely removing pathogenic alleles, regardless if they are dominant or recessive, obtaining permanently revised healthy cells. The rapid breakthrough in this technology and the existence of first academic clinical trial for cancer immunotherapies [126], render clinical application of CRISPR–Cas strategy in DOA a plausible objective, which will be certainly pursued soon. 4.2. Drugs potentially able to complement OPA1 defective function 4.2.1. Drugs targeting complex I and ROS In the last 10 years, our understanding of OPA1 mutations’ pathological effects has grown and, consequently, there has been an increasing interest in numerous molecules potentially able to treat their specific deleterious effects. The reduction of complex I dependent ATP synthesis observed in DOA fibroblasts [47] suggests the use of electron donors and acceptors, like coenzyme Q10 (CoQ10) and riboflavin, as potential drugs. The safety of CoQ10, an ubiquinone analogue, has been proven by its wide use in patients with various mitochondrial disorders. However, CoQ10 efficacy seems limited to a restricted category of diseases with a primary deficiency of this quinone [127]. The new generation of shorter chain analogues of ubiquinone, idebenone and EPI-743, was reported as potentially more effective than CoQ10 [17]. In a small trial the use of EPI-743 in five LHON patients, in a very early stage of the disease, induced some promising beneficial effects that await confirmation in a properly designed clinical trial [128]. Idebenone, widely used in treating neurodegenerative diseases, has been recently approved by the European Medicine Agency also for LHON [17,129,130]. Idebenone has a partial efficacy in LHON, depending on the disease stage and duration of therapy, as suggested by a large retrospective study involving 103 patients, where a greater proportion of treated patients recovered vision compared to the untreated group, and the early start of therapy was the most predictive factor for visual recovery [129]. This observation may explain the limited therapeutic effect of idebenone on RGCs dendropathy observed in the mouse model of DOA. Indeed, the 11-months-old mice showed a considerable improvement in visual acuity, although only for less than 6 weeks, whereas in 12to 14-months-old mice only a slight indication of benefit has been observed [131]. Encouraging results have been reported from a pilot open trial of idebenone treatment with DOA patients, where five of seven showed increased visual acuity and the improvements occurred after at least 6 months of idebenone administration. Also in this case, older patients with worst baseline visual acuity did not



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achieve improvement with therapy [132], supporting the necessity of an early-start treatment to obtain the best result. The link between OPA1 and increased ROS production [71,133] suggests the possible use of antioxidants, such as vitamins C, E, B2, B3, B12, lipoic acid and folic acid [17,134], to reduce the secondary toxic effect of OPA1 mutations. In most cases these supplements used to treat patients with different mitochondrial diseases lack any proven evidence of efficacy, and ROS are also involved in transducing signals in various pathways. Anyway, in a OPA1 Drosophila model with eye-specific somatic homozygous mutation, SOD1, vitamin E, and genetically overexpressed human SOD1 were all able to reverse the rough and glossy phenotype, suggesting that ROS may play an important role in cone and pigment cell death [67]. Moreover, Liu and colleagues highlighted the implication of ROS and mitochondrial defects in neurodegeneration in Drosophila and Ndufs4 mouse models, a pathological process that was significantly delayed by pharmacologic and genetic antioxidant treatments [135]. 4.2.2. Drugs modifying mitochondrial biogenesis and mitophagy The involvement of OPA1 in mtDNA content and stability [8,21] suggests a further therapeutic strategy based on drug-stimulated activation of mitochondrial biogenesis, such as with bezafibrate, rosiglitazone, resveratrol and AICAR, aimed at increasing gene transcription and respiratory chain complex activities. The efficacy of bezafibrate has been proven in vitro in fibroblasts and cybrids and in vivo in a muscle-specific PGC-1␣ transgenic mouse, although other studies did not confirm these positive results [134]. Treatment with AICAR increased differently respiratory chain complex activities in three models of COX deficiency, possibly dependent on the variable severity of the clinical phenotype [134]. Alternatively, altered autophagy and mitophagy caused by OPA1 mutation [27,90,91,93] may be a further intriguing therapeutic target. Rapamycin, a specific inhibitor of the mammalian target of rapamycin (mTOR), has been seen to delay onset of neurological symptoms in the Ndufs4-/- mouse by inducing a metabolic shift toward amino acid catabolism, away from glycolysis [136]. All these findings will need to be translated into OPA1 models to test the efficacy and feasibility of these possible future treatments.

Fig. 4. Summary of the therapeutic options to treat pathologies associated with OPA1 mutations. Mutations in OPA1 cause mitochondrial dysfunctions, responsible for RGCs degeneration. One of the new gene therapy (sky blue) is based on CRISPR-Cas9 system. This gene editing strategy allows the precisely removal of pathogenic alleles, blocking the process at the origin, whereas the gene therapy based on OPA1 is aimed in complementing the mutated allele, limiting the mitochondrial impairment. Mutations-independent strategies are developed to reduce RGCs degeneration, by inhibiting cell death or increasing neuronal survival. Pharmacological options (yellow) are designed to target a particular pathway or process damaged in mutated cells, to prevent the RGCs death. The figure was prepared following the guidelines for colorblind [139].

RGCs degeneration, encouraging future investigations and clinical trials for DOA patients [140]. Acknowledgements This research was supported by the FIR2013 grant RBFR131WDS to CZ, from the Ministero della Istruzione Università e Ricerca (MIUR). References

5. Conclusion Despite the growing knowledge on OPA1 functions and cellular pathways in which OPA1 is involved, an effective treatment for DOA is still missing. However, there is an expanding set of encouraging results from pre-clinical in vitro and animal studies, and some initial approaches in patients with drugs targeting mitochondrial dysfunction (Fig. 4). In particular, gene therapy holds promise to cure a large number of genetic diseases in the near future. There still remain some issues that need to be addressed and resolved in DOA and other neurodegenerative diseases. Complexity arises from the mutual dependence of RGCs and glial cells, such as oligodendrocytes and astrocytes, and their interaction has been shown to be crucial for axonal myelination and for unusual process like the recently described transmitophagy [137], a process where mitochondria are released from RGCs’ axons and degraded in the astrocytes [138]. It remains that gene therapy strategies, aimed at directing the OPA1 expression or replacement only within RGCs, may be not fully successful due to these complexities [138]. Further studies are needed to highlight the precisely pathogenic mechanism of DOA and related disorders: the development of new tools, such as organoids in combination with endonucleases, will be instrumental for testing present and future therapeutic approaches. In a recent paper, Lenaers and collegues showed the first indication that OPA1 gene therapy on a mouse model of DOA is able to alleviate

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